Potassium-conducting BK channels are attractive drug targets for the treatment of a wide variety of human disorders. Significant studies have shown that BK channels are distributed throughout the body and play important roles in a variety of physiological and disease processes. Compounds that inhibit or enhance BK channel function (modulators) have been tested or proposed to treat stroke, epilepsy, psychoses, pain, traumatic brain injury, hypokaleamic periodic paralysis, asthma, COPD, angina, arterial hypertension, ischemic heart disease, erectile dysfunction, urinary incontinence, gastric hypermotility, irritable bowel syndrome, pancreatitis, cancer (brain, bone, and prostate), rheumatoid arthritis, glaucoma and diabetes. However this diverse therapeutic potential remains largely untapped. Existing BK channel modulators have not advanced beyond clinical trials and suffer from poor potency and tissue specificity. Our long-term goal is to develop potent tissue-specific modulators of human BK channels. Such compounds should induce little to no adverse effects in non-target tissue, and therefore could serve as effective therapeutics as well as important research tools for the study of BK channel function in selected organs. Our previous research and preliminary results suggest that BK channel modulators exhibit diverse mechanisms of action which limit the ability of conventional high throughput screens to identify useful modulators. Therefore, the goal of this proposal is to develop and validate an innovative high throughput screening system, capable of detecting and evaluating the potency of small molecule modulators that act by different mechanisms of action and are selective for BK channel variants expressed in different tissues. The screen includes the novel use of mutant BK channels, engineered to increase the sensitivity of the assay to select modulators with the desired properties. The proposed screen will overcome several key barriers in the discovery and development of potent, tissue-specific BK channel modulators; and be suitable for screening hundreds of thousands of compounds.